Raman detection and imaging method and device based on solid optical microstructure

Abstract

The invention relates to the technical field of optical detection and imaging, and discloses a Raman detection and imaging method and device based on a solid optical microstructure. The method comprises the steps that S1, a solid optical microstructure is provided, the refractive index of the microstructure is different from that of a surrounding medium, so that incident exciting light is limited in a microcavity in the microstructure, and an enhancement area used for enhancing Raman scattering and self-fluorescence signals is formed at the interface of the microstructure; s2, positioning the microstructure, and enabling the enhanced area to be overlapped with the target detection area of the sample to be detected in space. S3, exciting light is coupled to the microstructure, the exciting light excites the target detection area of the to-be-detected sample through the enhancement area, and an enhanced Raman scattering signal and / or an autofluorescence signal are / is generated. And S4, acquiring the enhanced Raman scattering signal and / or self-fluorescence signal, and generating an image with a resolution below the optical diffraction limit based on the signal. Raman and self-fluorescence signals can be remarkably enhanced, super-resolution imaging is realized, and the detection sensitivity, resolution and stability are improved.

Description

Technical Field

[0001] The present application relates to the field of optical detection and imaging technology, and in particular to a Raman detection and imaging method and device based on solid optical microstructures. Background Art

[0002] This section is intended to provide a background or context for the embodiments of the present application as stated in the claims. The contents in this section are for reference only and do not constitute an admission or confirmation that they are prior art that has been disclosed.

[0003] When the excitation light interacts with molecules, inelastic scattering occurs, which is called Raman scattering. Under the action of the excitation light, the instantaneous dipole moment generated by the absorption of excitation energy by the molecules will have different characteristic Raman shifts due to different molecular structures. Therefore, the frequency and relative intensity of the Raman shift can be used for the qualitative and quantitative analysis of molecules, respectively. It has been widely used in non-labeled qualitative and quantitative detection and imaging in many fields such as materials, biology, chemistry, and environmental science. However, the excitation efficiency of spontaneous Raman scattering is only 10% of the incident photon. -6 Therefore, improving the intensity of Raman scattering has become the key to promoting the application of Raman scattering. In particular, the development of laser technology has enabled Raman scattering signals to be widely used.

[0004] With the recent development of confocal microscopy, Raman imaging, which uses Raman scattering signals to characterize the spatial distribution of molecules in a sample, has made significant progress. However, due to the low efficiency of spontaneous Raman scattering and the optical diffraction limit of optical microscopy, the maximum resolution of Raman imaging using confocal microscopy is limited to approximately 300 nanometers. Furthermore, due to the low excitation efficiency of Raman scattering, obtaining high signal-to-noise ratio and high-resolution Raman signal imaging often requires long spectral acquisition times or high excitation light powers, making Raman imaging unsuitable for biological samples. There are also reports on enhancing Raman scattering signals using techniques such as surface-enhanced Raman spectroscopy (SERS) and tip-enhanced Raman spectroscopy (TERS). However, these techniques require the presence of opaque metals, making them incompatible with optical microscopy imaging. Furthermore, metal-induced Raman scattering signal enhancement suffers from signal instability and poor reproducibility. Summary of the Invention

[0005] The purpose of this application is to provide a Raman detection and imaging method and device based on solid optical microstructures, which can significantly enhance Raman and autofluorescence signals and achieve super-resolution imaging through non-metallic solid optical microstructures, improve detection sensitivity, resolution and stability, and is suitable for a variety of microscopic imaging platforms.

[0006] This application discloses a Raman detection and imaging method based on solid optical microstructures, comprising the following steps:

[0007] S1. Providing a solid optical microstructure having a refractive index different from that of the surrounding medium, such that incident excitation light is confined to a microcavity within the microstructure and an enhancement region is formed at the interface of the microstructure for enhancing Raman scattering and autofluorescence signals;

[0008] S2. positioning the microstructure so that the enhanced region spatially overlaps with the target detection region of the sample to be tested;

[0009] S3. coupling excitation light to the microstructure, wherein the excitation light excites the target detection area of the sample to be tested through the enhancement region to generate an enhanced Raman scattering signal and / or autofluorescence signal;

[0010] S4. Collecting the enhanced Raman scattering signal and / or autofluorescence signal, and generating an image with a resolution below the optical diffraction limit based on the signal.

[0011] In a preferred embodiment, the invention further comprises:

[0012] The microstructure is controlled to generate relative displacement relative to the sample to be tested, and steps S2 to S4 are repeated to achieve the acquisition of the enhanced Raman scattering signal and / or autofluorescence signal from the multiple target detection areas of the sample to be tested, thereby constructing a two-dimensional or three-dimensional spectral image of the sample to be tested.

[0013] In a preferred embodiment, positioning the microstructure further comprises the following steps:

[0014] placing the microstructure on the surface of the target detection area of the sample to be tested;

[0015] Observing the microstructure using an objective lens, first adjusting the objective lens horizontally to find the microstructure, and then adjusting the objective lens vertically until a target image feature is observed;

[0016] Fine-tune the longitudinal distance by real-time spectroscopy until the strongest Raman signal is obtained;

[0017] Collect Raman spectra.

[0018] In a preferred embodiment, the enhancement region formed at the interface of the microstructure for enhancing Raman scattering and autofluorescence signals includes:

[0019] The microstructure forms an evanescent wave electric field on the surface; and

[0020] The excitation light passes through the microstructure to form a photon nanojet on a side away from the incident direction of the excitation light.

[0021] In a preferred example, the evanescent wave formed on the surface of the microstructure makes the Raman scattering and autofluorescence of the molecules on the surface of the microcavity more susceptible to excitation, thereby improving the sensitivity of Raman scattering and autofluorescence detection, and superimposing the photonic nanojet to achieve high-resolution Raman imaging below the optical diffraction limit.

[0022] In a preferred embodiment, the microcavity is formed by:

[0023] The excitation light is coupled into the interior of the solid optical microstructure, so that the excitation light undergoes total internal reflection at the interface between the microstructure and the surrounding medium, and continuously propagates along the inner wall of the microstructure, forming a stable optical resonance mode under predetermined structural size and wavelength conditions, thereby forming an optical microcavity with a high quality factor inside the microstructure.

[0024] In a preferred embodiment, positioning the microstructure further comprises:

[0025] The microstructure is placed above or below the target detection area of the sample to be tested, or the microstructure is directly placed on the surface of the target detection area of the sample to be tested and is in direct contact with the surface.

[0026] In a preferred embodiment, the solid optical microstructure is in the shape of a microcavity with a high quality factor, including a microsphere, a microring, a microcolumn or a microdisk.

[0027] In a preferred embodiment, the size of the solid optical microstructure is 5 μm-5 mm.

[0028] In a preferred embodiment, the solid optical microstructure is a microsphere, and the diameter of the microsphere is 10 μm-800 μm.

[0029] In a preferred embodiment, the enhanced Raman scattering signal and / or autofluorescence signal is collected by an optical detection device.

[0030] In a preferred embodiment, the optical detection device includes a charge coupled device, a photomultiplier tube or a photodiode.

[0031] In a preferred embodiment, the microstructure is also used to enhance optical signals generated based on stimulated emission mechanisms, including Raman scattering, autofluorescence, upconversion luminescence and / or photoluminescence signals.

[0032] In a preferred embodiment, the solid optical microstructure material is selected from inorganic glass or organic polymer material.

[0033] In a preferred embodiment, the inorganic glass is selected from the following group: borosilicate glass, soda lime glass, barium titanate glass, quartz glass, lead silicate glass or aluminosilicate glass.

[0034] In a preferred embodiment, the organic polymer material is selected from the following group: polystyrene, polymethyl acrylate or polyvinyl chloride.

[0035] In a preferred embodiment, the solid optical microstructure is used in conjunction with a Raman spectrometer, a fluorescence spectrometer, a Raman microscope or a fluorescence microscope to achieve high-resolution Raman imaging or autofluorescence imaging.

[0036] This application also discloses a Raman detection and imaging device based on a solid optical microstructure, comprising:

[0037] An excitation light path, used to provide excitation light to the sample area to be tested;

[0038] an objective lens, disposed in the excitation light path, for focusing the excitation light and collecting the spectral signal emitted by the sample to be tested;

[0039] A bracket is arranged below the objective lens;

[0040] a solid optical microstructure mounted on the support, wherein the refractive index of the solid optical microstructure is different from that of the surrounding medium, so that the incident excitation light is confined to the microcavity within the microstructure and forms an evanescent wave and / or a photon nanojet on the surface of the microstructure, thereby forming an enhanced region of Raman scattering and autofluorescence;

[0041] a sample stage, for supporting the sample to be tested, so that the enhancement zone and the target detection area of the sample to be tested spatially overlap, so that the target detection area of the sample to be tested is excited by the excitation light to generate an enhanced Raman scattering signal and / or autofluorescence signal;

[0042] A signal acquisition component is used to receive the enhanced Raman scattering signal and / or autofluorescence signal and generate image data.

[0043] In a preferred embodiment, a microhole is provided at the bottom of the bracket, and the diameter of the microhole is smaller than the maximum diameter of the microsphere, so that the microsphere is fixed in the microhole while the excitation light can be emitted through the microhole.

[0044] In a preferred embodiment, the center of the microstructure is located at the center of the objective lens.

[0045] This application has the following advantages:

[0046] The enhancement of Raman scattering in this application is based on the high Q value (high quality factor) of the dielectric microcavity. It does not require the addition of any additional equipment or the increase of the energy of the excitation light. The enhancement of the Raman scattering signal can be achieved only through the design of the optical device. In addition, the combination with the Raman microscope can break through the optical diffraction limit and construct an ultra-high-resolution Raman imaging technology, which can achieve an optical Raman imaging resolution of about 40 nanometers and can be widely used for high-resolution imaging of inorganic materials, tissues, cells and other biological samples. More importantly, this application can reduce the light damage of the laser to the sample, and is particularly suitable for biological samples that are more sensitive to heat (such as cells, tissues, etc.). Using a laser with a certain power, more friendly Raman high-resolution imaging can be obtained, which is of great value for achieving high-resolution fluorescence imaging of samples that use stimulated emission spectra and imaging such as autofluorescence.

[0047] The various technical features disclosed in the above invention content, the various technical features disclosed in the various embodiments and examples below, and the various technical features disclosed in the accompanying drawings can be freely combined with each other to form various new technical solutions (all of which should be deemed to have been recorded in this specification), unless such a combination of technical features is technically infeasible. For example, in one example, feature A+B+C is disclosed, and in another example, feature A+B+D+E is disclosed. Features C and D are equivalent technical means that play the same role. Technically, only one of them needs to be used, and it is impossible to use them at the same time. Feature E can be technically combined with feature C. In this case, the solution of A+B+C+D should not be deemed to have been recorded because it is technically infeasible, while the solution of A+B+C+E should be deemed to have been recorded. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is a flow chart of a Raman detection and imaging method based on solid optical microstructures according to the first embodiment of the present application.

[0049] Figure 2 This is a comparison chart of thermal sensitivities of mouse kidney tissue slices obtained by a high-resolution Raman imaging system according to one embodiment of the present application.

[0050] Figure 3 This is a Raman imaging diagram of a 42-nanometer-wide silicon stripe produced by a high-resolution Raman imaging system according to one embodiment of the present application.

[0051] Figure 4 This is a subcellular level Raman imaging diagram of an optical microsphere high-resolution Raman imaging system according to one embodiment of the present application.

[0052] Figure 5 This is a schematic diagram of an optical microsphere high-resolution Raman imaging system exploring the cell infection mechanism of viruses according to one embodiment of the present application.

[0053] Figure 6 It is a structural schematic diagram of a Raman detection device based on solid optical microstructure according to the second embodiment of the present application. DETAILED DESCRIPTION

[0054] In the following description, many technical details are provided to help readers better understand this application. However, those skilled in the art will understand that even without these technical details and various changes and modifications based on the following embodiments, the technical solutions claimed in this application can be implemented.

[0055] Description of some concepts:

[0056] High Quality Factor (Q): A physical quantity used to measure the relationship between energy storage and energy loss in an optical microcavity or resonant structure. It is typically defined as the ratio of the microcavity's resonant frequency to its spectral width at half maximum. A high Q indicates that light can propagate multiple times through the microcavity with minimal loss, resulting in a longer residence time and stronger resonance enhancement.

[0057] An evanescent wave is a non-propagating electromagnetic wave generated near an interface when light is totally reflected from one medium into another with a lower refractive index. This wave propagates along the interface but rapidly decays perpendicularly, with its energy concentrated in a subwavelength region near the interface.

[0058] Photonic nanojet: A focused beam of light with high intensity, low diffraction, subwavelength lateral dimensions, and long propagation distance is formed at the exit of a transparent microsphere or other dielectric microstructure whose size is close to or slightly larger than the wavelength. This beam is typically concentrated near the surface of the microstructure and exhibits extremely high spatial resolution, surpassing the diffraction limit of traditional optics and enabling precise excitation or detection of samples at the nanoscale.

[0059] The following is a summary of some of the innovative features of the embodiments of this application:

[0060] This application uses a solid optical microstructure with a different refractive index than the surrounding medium to form a dielectric microcavity structure with a high quality factor (high Q value) within the excitation light. This significantly enhances the Raman scattering signal without the need for additional metal reinforcement materials, additional equipment, or increased excitation light power. The excitation light is confined by the high-Q value microcavity and propagates repeatedly, forming a stable evanescent wave and / or photon nanojets on the microstructure surface, thus constructing an enhancement zone. This enhancement zone not only improves the excitation efficiency of Raman scattering, but also significantly increases the excitation intensity of stimulated emission signals such as autofluorescence.

[0061] In addition, since the optical enhancement mechanism adopted in this application is a pure dielectric structure, it avoids the problems of signal instability and poor biocompatibility that may be introduced by metal materials, significantly reduces the photothermal damage to the sample caused by the laser excitation process, and is particularly suitable for samples such as biological tissues, cells and proteins that are sensitive to lasers. By realizing Raman and / or fluorescence signal acquisition in the enhancement area, and then combining multi-point scanning with the relative displacement of the microstructure, two-dimensional or three-dimensional imaging results with high spatial resolution can be obtained, breaking through the resolution bottleneck of traditional Raman microscopy systems that are limited by the optical diffraction limit. Therefore, the technical solution proposed in this application not only has label-free, non-destructive, and highly sensitive Raman detection capabilities, but also has the potential to achieve super-resolution fluorescence imaging, providing a low-loss, highly compatible new imaging method for fields such as biomedical imaging, nanostructure analysis, and chemical detection.

[0062] In order to make the objectives, technical solutions and advantages of this application clearer, the implementation methods of this application will be further described in detail below with reference to the accompanying drawings.

[0063] The first embodiment of the present application relates to a Raman detection and imaging method based on solid optical microstructures, the process of which is as follows: Figure 1 As shown, the method includes the following steps:

[0064] S1. Providing a solid optical microstructure having a refractive index different from that of the surrounding medium, such that incident excitation light is confined to a microcavity within the microstructure and an enhancement region is formed at the interface of the microstructure for enhancing Raman scattering and autofluorescence signals;

[0065] S2. Positioning the microstructure so that the enhancement region spatially overlaps with the target detection region of the sample to be tested;

[0066] S3. coupling excitation light to the microstructure, the excitation light excites the target detection area of the sample to be tested through the enhancement zone to generate an enhanced Raman scattering signal and / or autofluorescence signal;

[0067] S4. Collecting the enhanced Raman scattering signal and / or the autofluorescence signal, and generating an image with a resolution below the optical diffraction limit based on the signal.

[0068] In an optional embodiment, the method may further include: controlling the microstructure to produce relative displacement relative to the sample to be tested, and then repeating steps S2 to S4 to achieve enhanced Raman scattering signal and / or autofluorescence signal collection for multiple target detection areas of the sample to be tested, thereby constructing a two-dimensional or three-dimensional spectral image of the sample to be tested.

[0069] In an optional embodiment, step S2 may further include the following steps:

[0070] The microstructure is placed on the surface of the target detection area of the sample to be tested.

[0071] Use the objective lens to observe the microstructure. First, adjust the objective lens horizontally to find the microstructure, and then adjust the objective lens vertically until the target image feature is observed.

[0072] The longitudinal distance was fine-tuned by real-time spectroscopy until the strongest Raman signal was obtained.

[0073] Collect Raman spectra.

[0074] In an optional embodiment, forming an enhancement region at the interface of the microstructure for enhancing Raman scattering and autofluorescence signals includes:

[0075] The microstructure creates an evanescent electric field on the surface; and,

[0076] The excitation light passes through the microstructure and forms a photon nanojet on the side away from the incident direction of the excitation light.

[0077] In an optional embodiment, the evanescent wave formed on the microstructure surface makes the Raman scattering and autofluorescence of the molecules on the microcavity surface more susceptible to excitation, thereby improving the sensitivity of Raman scattering and autofluorescence detection, and superimposing photonic nanojets to achieve high-resolution Raman imaging below the optical diffraction limit.

[0078] In an optional embodiment, the target image feature may include target tissue or diffraction spot, etc.

[0079] In an optional embodiment, the microcavity is formed by coupling excitation light into the interior of a solid optical microstructure, causing the excitation light to undergo total internal reflection at the interface between the microstructure and the surrounding medium, and continuously propagating along the inner wall of the microstructure, forming a stable optical resonance mode under predetermined structural size and wavelength conditions, thereby forming an optical microcavity with a high quality factor inside the microstructure.

[0080] In an optional embodiment, positioning the microstructure further includes placing the microstructure above, below, or directly contacting the surface of the target detection area of the sample to be tested.

[0081] In an optional embodiment, the solid optical microstructure is in the shape of a microcavity with a high quality factor, including a microsphere, a microring, a micropillar, or a microdisk.

[0082] In an optional embodiment, the size of the solid optical microstructure is 5 μm-5 mm.

[0083] In an optional embodiment, the solid optical microstructure is a microsphere, and the diameter of the microsphere is 10 μm-800 μm.

[0084] In an optional embodiment, the enhanced Raman scattering signal and / or the autofluorescence signal is collected by an optical detection device.

[0085] In an optional embodiment, the optical detection device includes a charge coupled device, a photomultiplier tube or a photodiode.

[0086] In an optional embodiment, the microstructure is further used to enhance optical signals generated based on stimulated emission mechanisms, including Raman scattering, autofluorescence, upconversion luminescence and / or photoluminescence signals.

[0087] In an optional embodiment, the solid optical microstructure material is selected from inorganic glass or organic polymer material.

[0088] In an optional embodiment, the inorganic glass is selected from the group consisting of borosilicate glass, soda lime glass, barium titanate glass, quartz glass, lead silicate glass, or aluminosilicate glass.

[0089] In an optional embodiment, the organic polymer material is selected from the following group: polystyrene, polymethyl acrylate or polyvinyl chloride.

[0090] In an optional embodiment, the solid optical microstructure is used in conjunction with a Raman spectrometer, a fluorescence spectrometer, a Raman microscope, or a fluorescence microscope to achieve high-resolution Raman imaging or autofluorescence imaging.

[0091] In order to better understand the technical solution of the present application, a specific example is provided below for illustration. The details listed in the example are mainly for ease of understanding and are not intended to limit the scope of protection of the present application.

[0092] Example 1: Optical microspheres enhance the Raman spectroscopy signal of single-crystal silicon

[0093] Place K9 glass microspheres (10-1000 microns in diameter) with uniform particle size and smooth surface on a special accessory (such as a bracket) and observe after matching it with a 10× objective lens. First, adjust the objective lens in the horizontal position to find the microspheres, then adjust the longitudinal position of the objective lens until the 0th order diffraction spot is observed, and use the real-time spectrum to fine-tune the longitudinal distance until the strongest Raman signal is obtained, and collect the Raman spectrum. The spectrum collection parameters are as follows: the excitation wavelength is selected as 532 nanometers, the laser power is 10 milliwatts, the integration time is 3 seconds, and the cumulative number of times is 10 times. The quantitative basis is to select the characteristic Raman peak of single crystal silicon, the peak of the Si-Si bond is located at 520 cm -1 Experimental results show that the Raman signal of single crystal silicon can be enhanced by 11 times or more when optical microspheres are present.

[0094] Example 2: Optical microspheres enhance tissue Raman spectroscopy signals

[0095] Fresh mouse kidney tissue was fixed or directly cut to the required size. OCT (Optimal Cutting Temperature compound) cryomounts were prepared in a -80°C refrigerator. When the OCT was close to solidifying, the mouse kidney tissue was quickly placed in the semi-solidified OCT, then embedded in OCT, and finally stored in a -80°C refrigerator. When slicing, the quick-frozen tissue was placed at room temperature for about 1 minute. After the tissue was warmed to -20°C, the slices were sliced. The slides were placed at room temperature, and the slices were placed flat on the slides and fixed with OCT. The slices were washed 3-5 times with PBS (Phosphate Buffered Saline) buffer solution for 5 minutes each time to remove OCT and prevent spectral interference.

[0096] Then the Raman test of this application was carried out, and K9 glass microspheres (diameter 10-1000 microns) with uniform particle size and smooth surface were placed on the surface of mouse kidney tissue and observed with a 10× objective lens. First, adjust the objective lens in a horizontal position to find the microspheres, and then adjust the longitudinal position of the objective lens until the tissue is observed. Use the real-time spectrum to fine-tune the longitudinal distance until the strongest Raman signal is obtained, and collect the Raman spectrum. The acquisition parameters of the spectrum are as follows: the excitation wavelength is selected as 532 nanometers, the laser power is 20 milliwatts, the integration time is 10 seconds, and the cumulative number of times is 10 times. The quantitative basis is to select the tissue Raman shifts at 1446, 1658 and 2933 cm -1 The Raman signals of tissues can be enhanced by at least 2-4 times when optical microspheres are present.

[0097] Example 3 Optical microspheres enhance Raman signal with low thermal damage

[0098] K9 glass microspheres (10-1000 microns in diameter) with uniform particle size and smooth surface are placed on the surface of mouse kidney tissue and observed through an objective lens. First, adjust the objective lens in a horizontal position to find the microspheres, then adjust the longitudinal position of the objective lens until the tissue is observed, and use the real-time spectrum to fine-tune the longitudinal distance until the strongest Raman signal is obtained, and collect the Raman spectrum. The laser irradiates the tissue, and the Raman spectrum is collected every 3 minutes. The 40× objective lens and the scene where the microstructure is placed are measured 10 times each. The spectrum acquisition parameters are an excitation wavelength of 532 nanometers, a laser power of 20 milliwatts, an integration time of 10 seconds, and a cumulative number of 10 times. Figure 2Figure 1 shows a comparison of thermal sensitivity obtained by a high-resolution Raman imaging system on mouse kidney tissue slices. Figure a shows a series of Raman imaging images of a mouse kidney slice using the dielectric microsphere-based high-resolution Raman imaging technology of this application, which has high resolution and good imaging time tolerance. Figure b shows a series of Raman imaging images of a mouse kidney tissue slice without the dielectric microspheres of this application. The experimental results show that when the optical microspheres are present, thermal damage to the tissue can be reduced by 2.5 times or more.

[0099] Example 4: Optical Microsphere Enhanced Raman Imaging for Tissue Imaging

[0100] First, for tissue and cell imaging, the recommended signal-to-noise ratio is: the signal-to-noise ratio of the CH peak reaches above 12. The higher the signal-to-noise ratio, the better, but the overall imaging time and sample power tolerance need to be taken into account. K9 glass microspheres (diameter 10-1000 microns) with uniform particle size and smooth surface are placed on the surface of mouse kidney tissue and observed using an objective lens. First, adjust the objective lens in a horizontal position to find the microspheres, then adjust the longitudinal position of the objective lens until the tissue is observed, and use real-time spectroscopy to fine-tune the longitudinal distance until the strongest Raman signal is obtained, and collect Raman spectra. The spectrum acquisition parameters are as follows: the excitation wavelength is selected as 532 nanometers, the laser power is 20 milliwatts, and the accumulation number is 10 times. Set different integration times (0.5, 1, 1.5, 2, 3, 4, 6, 8, and 10 seconds), collect single spectra and calculate the signal-to-noise ratio. Select the appropriate integration time and use a confocal Raman microscope to image the tissue. K9 glass microspheres (10-1000 microns in diameter) with uniform particle size and smooth surface were placed on the surface of mouse kidney tissue. The tissue was observed using an objective lens, with imaging parameters ranging from 1×1 to 20×20 microns and a step size of 20-500 nanometers. Spectral acquisition parameters included an excitation wavelength of 532 nanometers, a laser power of 20 milliwatts, an integration time of 4 seconds, and 10 accumulations. The experimental results demonstrated the successful implementation of Raman imaging of tissue using optical microsphere-enhanced Raman spectroscopy.

[0101] Example 5 High-resolution Raman imaging of optical microspheres

[0102] K9 glass microspheres (10-1000 microns in diameter) with uniform particle size and smooth surface are placed on special accessories (such as a bracket) and immersed in water, and then matched with a 63× water immersion objective lens for observation. First, adjust the objective lens in a horizontal position to find the microspheres, and then adjust the longitudinal position of the objective lens until the diffraction spot is observed. Use the real-time spectrum to fine-tune the longitudinal distance until the strongest Raman signal is obtained, and determine it as the focal plane for imaging. The acquisition area is 4×4 microns, with a step size of 20 nanometers. The acquisition parameters of the spectrum are an excitation wavelength of 532 nanometers, a laser power of 20 milliwatts, and an integration time of 0.01 seconds. The characteristic Raman peak of single-crystalline silicon is selected for imaging, and the peak of the Si-Si bond is located at 520 cm -1The experimental results show that when the optical microspheres of the present application exist, the optical diffraction limit can be broken through, and Raman imaging at the nanoscale can be achieved with a resolution of about 40 nanometers. Figure 3 The figure shows the Raman imaging of 42 nanometer wide silicon stripes by the high-resolution Raman imaging system. Figure 3 A in the figure is the atomic force microscopy (AFM) imaging of silicon stripes; Figure 3 Figure B is the signal intensity distribution diagram of AFM imaging of silicon stripes; Figure 3 C in FIG. 1 is a Raman image of a silicon stripe pattern obtained by using the optical microsphere super-resolution Raman imaging technology of the present application; Figure 3 D in the figure is the signal intensity distribution diagram of high-resolution Raman imaging (Raman) of silicon stripes.

[0103] Example 6: Raman imaging at the subcellular level using high-resolution imaging of optical microspheres

[0104] K9 glass microspheres (10-1000 microns in diameter) with uniform particle size and smooth surface are placed on special accessories (such as a bracket) and immersed in water, and then matched with a 63× water immersion objective lens for observation. First, adjust the objective lens in a horizontal position to find the microspheres, and then adjust the longitudinal position of the objective lens until the diffraction spot is observed. Use the real-time spectrum to fine-tune the longitudinal distance until the strongest Raman signal is obtained, and determine it as the focal plane of the imaging. The acquisition area is 4×4 microns, and the step length is 20 nanometers. The acquisition parameters of the spectrum are an excitation wavelength of 532 nanometers, a laser power of 20 milliwatts, an integration time of 2 seconds, and a cumulative number of 10 times. The experimental results show that the use of optical microspheres to enhance Raman high-resolution imaging achieves clear Raman imaging of the nucleolus, nucleoplasm and nuclear membrane in the cell nucleus, with a resolution of about 40 nanometers, such as Figure 4 Shown is subcellular Raman imaging of the optical microsphere high-resolution Raman imaging system. The Raman imaging of the nucleus of A549 cells (left) (the range marked by the red frame) is compared using the non-microsphere Raman high-resolution imaging technology (upper right) and the microsphere Raman high-resolution imaging technology (lower right). It can be seen that the Raman imaging resolution of the nucleus of A549 cells (left) using the microsphere Raman high-resolution imaging technology (lower right) is higher.

[0105] Example 7: Observation of viral infection mechanism in cells using high-resolution Raman imaging of optical microspheres

[0106] K9 glass microspheres (10-1000 microns in diameter) with uniform particle size and smooth surface are placed on special accessories (such as brackets) and immersed in water, and then matched with a 63× water immersion objective lens for observation. First, adjust the objective lens in a horizontal position to find the microspheres, and then adjust the longitudinal position of the objective lens until the diffraction spot is observed. Use the real-time spectrum to fine-tune the longitudinal distance until the strongest Raman signal is obtained, and determine it as the focal plane of the imaging. The acquisition area is 4×4 microns, and the step length is 20 nanometers. The acquisition parameters of the spectrum are an excitation wavelength of 532 nanometers, a laser power of 20 milliwatts, an integration time of 2 seconds, and a cumulative number of 10 times. The experimental results show that the use of optical microspheres to enhance Raman high-resolution imaging successfully achieved the formation of intercellular ducts in A549 cells infected with influenza virus, as well as the transmission of the virus. After the virus infects the host cell, it uses the infected cells to replicate and proliferate itself, and induces the formation of ducts between cells. The virus is transmitted from one cell to another through the intercellular ducts, expanding the infection. As Figure 5 The figure shows the exploration of the virus cell infection mechanism by the optical microsphere high-resolution Raman imaging system. The microsphere high-resolution Raman imaging technology of this application (right) is used to image A549 cells infected with influenza virus. Raman imaging can be performed on the intercellular ducts (marked by the red line). The red clumps are lipid methylene groups (2850 cm -1 ) and the unsaturated double bonds of viral lipids (3010 cm -1 )’s Raman signal distribution, and the Raman imaging resolution is about 40 nanometers.

[0107] The second embodiment of the present application relates to a Raman detection and imaging device based on a solid optical microstructure, the structure of which is as follows: Figure 6 As shown, the Raman detection and imaging device based on solid optical microstructure includes: an excitation light path, an objective lens, a bracket, a solid optical microstructure, a sample stage and a signal acquisition component.

[0108] The excitation light path is used to provide excitation light to the sample area to be measured.

[0109] The objective lens is arranged in the excitation light path and is used to focus the excitation light and collect the spectral signal emitted by the sample to be tested.

[0110] The bracket is arranged below the objective lens.

[0111] The solid optical microstructure is mounted on a bracket. The refractive index of the solid optical microstructure is different from that of the surrounding medium, so that the incident excitation light is confined to the microcavity within the microstructure and forms evanescent waves and / or photon nanojets on the surface of the microstructure to form an enhanced area of Raman scattering and autofluorescence.

[0112] The sample stage is used to support the sample to be tested, so that the enhancement zone and the target detection area of the sample to be tested overlap in space, so that the target detection area of the sample to be tested is excited by the excitation light to produce enhanced Raman scattering signals and / or autofluorescence signals.

[0113] The signal acquisition component is used to receive the enhanced Raman scattering signal and / or the autofluorescence signal and generate image data.

[0114] In an optional embodiment, a microhole is provided at the bottom of the bracket, and the diameter of the microhole is smaller than the maximum diameter of the microsphere, so that the microsphere is fixed in the microhole while the excitation light can be emitted through the microhole.

[0115] In an optional embodiment, the center of the microstructure is located at the center of the objective lens.

[0116] The first embodiment is a method embodiment corresponding to the present embodiment. The technical details in the first embodiment can be applied to the present embodiment, and the technical details in the present embodiment can also be applied to the first embodiment.

[0117] It should be noted that, in this application, relational terms such as first and second are merely used to distinguish one entity or operation from another, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprise," "include," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article, or device. Without further restriction, an element defined by the phrase "comprising a" does not exclude the presence of other identical elements in the process, method, article, or device comprising the element. In this application, if it is mentioned that an action is performed according to a certain element, it means that the action is performed at least according to that element, including two situations: performing the action only according to that element, and performing the action according to that element and other elements. Expressions such as "multiple," "multiple," and "multiple" include 2, 2 times, 2 kinds, and more than 2, more than 2 times, and more than 2 kinds.

[0118] The serial numbers used in describing the steps of a method do not themselves limit the order of these steps. For example, a step with a larger serial number does not necessarily have to be executed after a step with a smaller serial number. The step with a larger serial number can be executed first and then the step with a smaller serial number, or they can be executed in parallel, as long as this execution order is reasonable to those skilled in the art. For another example, having multiple steps with consecutive serial numbers (e.g., step 101, step 102, step 103, etc.) does not limit other steps that can be executed in between. For example, there can be other steps between step 101 and step 102.

[0119] This specification includes combinations of the various embodiments described herein. Individual references to embodiments (e.g., "one embodiment" or "some embodiments" or "preferred embodiments") are not mutually exclusive unless indicated as such or clear to one skilled in the art. It should be noted that the word "or" is used in this specification in a non-exclusive sense unless the context clearly indicates or requires otherwise.

[0120] All documents mentioned in this specification are considered to be included in their entirety in the disclosure of this application so that they can be used as a basis for modification when necessary. In addition, it should be understood that the above description is only a preferred embodiment of this specification and is not intended to limit the scope of protection of this specification. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of this specification should be included in the scope of protection of one or more embodiments of this specification.

Claims

1. A Raman detection and imaging method based on solid optical microstructures, characterized in that: The steps include: S1. Providing a solid optical microstructure having a refractive index different from that of the surrounding medium, such that incident excitation light is confined to a microcavity within the microstructure and an enhancement region is formed at the interface of the microstructure for enhancing Raman scattering and autofluorescence signals; S2. positioning the microstructure so that the enhanced region spatially overlaps with the target detection region of the sample to be tested; S3. coupling excitation light to the microstructure, wherein the excitation light excites the target detection area of the sample to be tested through the enhancement region to generate an enhanced Raman scattering signal and / or autofluorescence signal; S4. Collecting the enhanced Raman scattering signal and / or autofluorescence signal, and generating an image with a resolution below the optical diffraction limit based on the signal.

2. The Raman detection and imaging method based on solid optical microstructures according to claim 1, characterized in that: Further including: The microstructure is controlled to generate relative displacement relative to the sample to be tested, and steps S2 to S4 are repeated to achieve the acquisition of the enhanced Raman scattering signal and / or autofluorescence signal from the multiple target detection areas of the sample to be tested, thereby constructing a two-dimensional or three-dimensional spectral image of the sample to be tested. In a preferred embodiment, positioning the microstructure further comprises the following steps: placing the microstructure on the surface of the target detection area of the sample to be tested; Observing the microstructure using an objective lens, first adjusting the objective lens horizontally to find the microstructure, and then adjusting the objective lens vertically until a target image feature is observed; Fine-tune the longitudinal distance by real-time spectroscopy until the strongest Raman signal is obtained; Collect Raman spectra. In a preferred embodiment, the enhancement region formed at the interface of the microstructure for enhancing Raman scattering and autofluorescence signals includes: The microstructure forms an evanescent wave electric field on the surface; as well as, The excitation light passes through the microstructure to form a photon nanojet on a side away from the incident direction of the excitation light. In a preferred example, the evanescent wave formed on the surface of the microstructure makes the Raman scattering and autofluorescence of the molecules on the surface of the microcavity more susceptible to excitation, thereby improving the sensitivity of Raman scattering and autofluorescence detection, and superimposing the photonic nanojet to achieve high-resolution Raman imaging below the optical diffraction limit.

3. The Raman detection and imaging method based on solid optical microstructures according to claim 1, characterized in that: The microcavity is formed by: The excitation light is coupled into the interior of the solid optical microstructure, so that the excitation light undergoes total internal reflection at the interface between the microstructure and the surrounding medium, and continuously propagates along the inner wall of the microstructure, forming a stable optical resonance mode under predetermined structural size and wavelength conditions, thereby forming an optical microcavity with a high quality factor inside the microstructure.

4. The Raman detection and imaging method based on solid optical microstructures according to claim 1, wherein: Positioning the microstructure further comprises: The microstructure is placed above or below the target detection area of the sample to be tested, or the microstructure is directly placed on the surface of the target detection area of the sample to be tested and is in direct contact with the surface.

5. The Raman detection and imaging method based on solid optical microstructures according to claim 1, characterized in that: The solid optical microstructure is in the shape of a microcavity with a high quality factor, including a microsphere, a microring, a microcolumn or a microdisk. In a preferred embodiment, the diameter of the solid optical microstructure is 5 μm-5 mm. In a preferred embodiment, the solid optical microstructure is a microsphere, and the diameter of the microsphere is 10 μm-800 μm.

6. The Raman detection and imaging method based on solid optical microstructures according to claim 1, wherein: The enhanced Raman scattering signal and / or autofluorescence signal is collected by an optical detection device. In a preferred embodiment, the optical detection device includes a charge coupled device, a photomultiplier tube or a photodiode.

7. The Raman detection and imaging method based on solid optical microstructures according to claim 1, characterized in that: The microstructures are also used to enhance optical signals generated based on stimulated emission mechanisms, including Raman scattering, autofluorescence, upconversion luminescence and / or photoluminescence signals.

8. The Raman detection and imaging method based on solid optical microstructures according to claim 1, wherein: The solid optical microstructure material is selected from inorganic glass or organic polymer material. In a preferred embodiment, the inorganic glass is selected from the following group: borosilicate glass, soda lime glass, barium titanate glass, quartz glass, lead silicate glass or aluminosilicate glass. In a preferred embodiment, the organic polymer material is selected from the following group: polystyrene, polymethyl acrylate or polyvinyl chloride.

9. The Raman detection and imaging method based on solid optical microstructures according to claim 1, wherein: The solid optical microstructure is used in conjunction with a Raman spectrometer, a fluorescence spectrometer, a Raman microscope or a fluorescence microscope to achieve high-resolution Raman imaging or autofluorescence imaging.

10. A Raman detection and imaging device based on solid optical microstructures, characterized in that: include: An excitation light path, used to provide excitation light to the sample area to be tested; an objective lens, disposed in the excitation light path, for focusing the excitation light and collecting the spectral signal emitted by the sample to be tested; A bracket is arranged below the objective lens; a solid optical microstructure mounted on the support, wherein the refractive index of the solid optical microstructure is different from that of the surrounding medium, so that the incident excitation light is confined to the microcavity within the microstructure and forms an evanescent wave and / or a photon nanojet on the surface of the microstructure, thereby forming an enhanced region of Raman scattering and autofluorescence; a sample stage, for supporting the sample to be tested, so that the enhancement zone and the target detection area of the sample to be tested spatially overlap, so that the target detection area of the sample to be tested is excited by the excitation light to generate an enhanced Raman scattering signal and / or autofluorescence signal; A signal acquisition component is used to receive the enhanced Raman scattering signal and / or autofluorescence signal and generate image data. In a preferred embodiment, a microhole is provided at the bottom of the bracket, and the diameter of the microhole is smaller than the maximum diameter of the microsphere, so that the microsphere is fixed in the microhole while the excitation light can be emitted through the microhole. In a preferred embodiment, the center of the microstructure is located at the center of the objective lens.

Images